The significance of fungal infections has been grossly underestimated. Only a few drugs are clinically available to treat life-threatening fungal infections, and resistance against these drugs is rising. Antifungal peptides (AFPs) are being actively explored as novel pharmaceuticals. PAF26 is a de novo designed hexapeptide possessing N-terminal cationic and C-terminal hydrophobic regions. Previously the roles of each of these motifs in the antifungal mode-of-action of PAF26 have indicated that it involves three stages: interaction with the plasma membrane, internalisation, and cell killing. The overall aim of my project was to obtain further insights into its mode-of-action and develop more active antifungal derivatives of PAF26. Three experimental fungal systems were used in this study: the model Neurospora crassa, the human pathogen Aspergillus fumigatus and the plant/human pathogen Fusarium oxysporum. The first objective of the study was to evaluate the impact of different fluorescent labels on the intracellular localisation and antifungal properties of PAF26. For this purpose a library of PAF26 labelled with 13 different fluorophores was synthesised. This library contained PAF26 conjugates of broad chemical and spectral diversity. These fluorescent PAF26 conjugates were analysed by live-cell imaging and tested for their antifungal activities. The different fluorescent labels were found to have significant impacts both on intracellular localisation and antifungal activities. TMR, carboxyfluorescein, NBD and DMN were found to be the best labels for live-cell imaging because they had the least influence on the intracellular localisation and antifungal activity of PAF26. The second objective was to identify target proteins of PAF26 in N. crassa cells. A large number of proteins were identified as binding to PAF26 from a protein pull-down and mass spectroscopy analysis using TMR- and fluorescein-labelled PAF26. One of these proteins was the highly abundant plasma membrane ATPase PMA-1. An in-silico analysis showed that PMA-1 is likely to be a major target protein of PAF26. The final objective was to develop novel antifungals based on PAF26 with improved activities and stability. Novel cyclic derivatives of PAF26 were designed in-silico against PMA-1. These peptides were synthesised and tested against N. crassa, A. fumigatus and F. oxysporum and were found to have higher activities (at the sub-micromolar level) and greater stability than the linear PAF26. Overall this study has provided novel mechanistic insights into the mode-of-action of PAF26 and discovered novel highly active antifungal peptides with clinical potential as therapeutics.

Liu, Yang. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 99-104). / Abstracts in English and Chinese. / Table of Contents / Thesis/Assessment Committee --- p.ii / Statement --- p.iii / Abstract --- p.iv / 摘要 --- p.vi / Acknowledgements --- p.vii / Table of Contents --- p.viii / List of Tables --- p.xi / List of Figures --- p.xii / List of Abbreviations --- p.xiv / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1. --- The plant secretory pathway --- p.2 / Chapter 2. --- Vesicular pathways in plant cells --- p.3 / Chapter 3. --- Prevacuolar Compartments --- p.6 / Chapter 4. --- Vacuolar sorting receptors (VSRs) --- p.7 / Chapter 5. --- BP-80 & Arabidopsis VSR Proteins --- p.8 / Chapter 6. --- Research Objectives --- p.9 / Chapter Chapter 2 --- Development and Expression of GFP-OsVSRs Fusion Reporters in Tobacco BY-2 and Rice Suspension Cultured Cells --- p.11 / Chapter 1. --- Introduction --- p.12 / Chapter 2. --- Materials and methods --- p.14 / Chapter 2.1 --- Construction of GFP-OsVSR chimeric reporters --- p.14 / Chapter 2.2 --- Construction of Golgi Marker and PVC marker --- p.18 / Chapter 2.3 --- Agrobacterium electroporation --- p.27 / Chapter 2.4 --- Transformation of tobacco BY-2 cells --- p.27 / Chapter 2.5 --- Transient expression of GFP-OsVSRs in protoplasts of tobacco BY-2 cells and rice suspension cultured cells --- p.28 / Chapter 2.6 --- Screening of transgenic BY-2 cells expressing GFP-OsVSR reporters --- p.30 / Chapter 2.7 --- Chemicals --- p.33 / Chapter 3. --- Results --- p.34 / Chapter 3.1 --- Study subcellular localization of OsVSR proteins with chimeric GFP-OsVSR reporters --- p.34 / Chapter 3.2 --- Generation of transgenic tobacco BY-2 cell lines expressing GFP-OsVSR reporter constructs --- p.38 / Chapter 3.3 --- Transient expression of GFP-OsVSR reporters in tobacco BY-2 and rice cell protoplasts --- p.40 / Chapter 4. --- Conclusion --- p.42 / Chapter Chapter 3 --- Subcellular Localization of GFP-OsVSR Fusion Reporters in Tobacco BY-2 and Rice Suspension Cultured Cells --- p.43 / Chapter 1. --- Introduction --- p.44 / Chapter 2. --- Materials and methods --- p.45 / Chapter 2.1 --- Confocal immunofluorescence studies --- p.45 / Chapter 2.2 --- Antibodies --- p.46 / Chapter 2.3 --- Wortmannin and BFA drug treatment --- p.46 / Chapter 2.4 --- Electron microscopy of resin-embedded cells --- p.47 / Chapter 2.5 --- Two-dimensional (2-D) gel analysis --- p.47 / Chapter 3 --- Results --- p.49 / Chapter 3.1 --- "Distinct subcellular localizations of GFP-OsVSRl, GFP-OsVSR2 and GFP-OsVSR4 reporters in transgenic BY-2 cell lines" --- p.49 / Chapter 3.2 --- "Subcellular localizations of GFP-OsVSRl, GFP-OsVSR2 and GFP-OsVSR4 in protoplasts of rice suspension cultured cells" --- p.58 / Chapter 3.3 --- Distinct localizations of GFP-OsVSR3 and GFP-OsVSR5 --- p.62 / Chapter 3.4 --- Immunogold EM localization of VSR proteins in rice suspension cultured cells --- p.65 / Chapter 3.5 --- 2-D western blot detection of VSR proteins in various plants --- p.68 / Chapter 4. --- Conclusions --- p.70 / Chapter Chapter 4 --- Summary and Discussion --- p.71 / Chapter 1. --- The significance of this study --- p.72 / Chapter 2. --- The hypothesis in this study --- p.73 / Chapter 3. --- A reporter system to study subcellular localization of OsVSR proteins in both tobacco BY-2 cells and rice suspension cultured cells --- p.75 / Chapter 4. --- Transiently expression of GFP-OsVSR reporters in BY-2 and rice protoplasts ..… --- p.76 / Chapter 5. --- Distinct PVC and Golgi localizations of GFP-OsVSR fusions --- p.77 / Chapter 6. --- Summary and future perspective --- p.78 / Appendix --- p.79 / Characterization of A Novel Rice Protein --- p.79 / Chapter 1. --- Introduction --- p.80 / Chapter 2. --- Materials and methods --- p.83 / Chapter 2.1 --- Antibodies --- p.83 / Chapter 2.2 --- Sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) and western blot analysis of proteins from different plant species --- p.84 / Chapter 2.3 --- Sucrose gradient fractionation with protein F antibody --- p.84 / Chapter 2.4 --- Confocal immunofluorescence studies --- p.85 / Chapter 2.5 --- Affinity purification of Protein F by its antibody --- p.85 / Chapter 3. --- Results --- p.87 / Chapter 3.1 --- Protein F is presented in different plant species --- p.87 / Chapter 3.2 --- Protein F is an integral membrane protein --- p.89 / Chapter 3.3 --- Subcelluar localization of Protein F --- p.91 / Chapter 3.4 --- Affinity purification of Protein F for identification --- p.95 / Chapter 4. --- Summary and future perspectives --- p.97 / Chapter 4.1 --- Summary --- p.97 / Chapter 4.2 --- Future Perspectives --- p.97 / References --- p.99

The development of new drug therapies traditionally requires mass screening of thousands if not millions of substances to identify lead compounds. They are then further optimised to increase potency. The screening of the large pharmaceutical compound libraries can be incredibly expensive, with the industry responding by miniaturising the assays to smaller formats, enabling the compound screening to be automated and, importantly, eliminating assay reagents that are a major contributing cost for running large screens. A potential target for such an approach is the genetic recoding site of viruses like HIV-1 and SARS. They use programmed recoding of the genetic code to regulate the translation of necessary proteins required for viable virus production. For example HIV-1 uses a -1 frameshift mechanism to regulate the ratio of the Gag to the Pol proteins, crucial for viable virus formation. The study of recoding, including readthrough of premature termination codons have most recently used bicistronic reporters with different combinations of enzymes. The most widely used plasmid bicistronic reporter utilises a dual luciferase arrangement comprised of firefly luciferase and Renilla luciferase reporters flanking the DNA being studied. Both of the luciferase enzymatic reporters emit light in response to their respective substrates. The cost of these substrates is the major issue to using luciferase reporters for high throughput screening. My study aimed at designing and developing a bicistronic assay suitable for genetic recoding that was amenable to high throughput screening. The luciferase reporters were replaced with Green Fluorescent Protein (GFP) reporters that do not require the addition of substrates. The development of a dual GFP assay required the appropriate selection of GFP fluorophores, the best arrangement of the GFPs to maximise the ratio of relative fluorescence intensity signal to background, the optimisation of the cells and growth conditions, DNA transfection, plate reader selection, and optical filter sets. Cassettes encoding protein linkers were also incorporated into the design of the constructs to separate the fluorescent proteins spatially to facilitate unimpaired folding into their functional units within the fusion protein. The assay was further improved by moving from transient transfection to stably expressing cell lines. A viable assay was almost achieved for 96 (and 384) well plates with a Z� factor compatible with the assay being suitable for high throughput screening. The assay was used to test a small collection of compounds known to interact with the ribosome and compounds known in the literature to affect frameshifting. This proof of concept was important, since it showed that the assay, with the various modifications, optimisations and miniaturisation steps, still retained the capability of correctly measuring the -1 frameshifting efficiency at the HIV-1 recoding site, and recording compound-induced modulations to the frameshifting efficiency. The compounds cycloheximide and anisomycin, for example, were shown to decrease -1 frameshifting albeit at some expense to overall protein synthesis. The dual GFP assay was also shown to be able to measure accurately changes in the frameshift efficiency brought about by mutations to the frameshift element, and additionally, it would be suitable for the detection and study of compounds, like the recently reported PTC-124 (currently undergoing phase II clinical trial for Duchenne Muscular Dystrophy and cystic fibrosis) that increases readthrough of a UGA premature stop codon mutation. The dual GFP assay developed in this study is at most only 1/10th of the cost of a comparable dual luciferase assay, largely due to removal of assay substrates and transfection reagents. The assay has a robust Z� factor comparable to that of the dual luciferase assay, and would substantially decrease the costs of high throughput screening in situations where a bicistronic reporter is required. The HIV-1 frameshift element is such a site.

Fluorescent analogs of biomolecules have been known as useful probes to study the structure, conformations and dynamics of cellular processes. These probes are more ideal than fluorescent labeled probes, as fluorescent analog probes retain the shape, size, conformation, and recognition element of the natural substrate, while giving useful intracellular information about detection and dynamics of biomolecules. The monoamine neurotransmitters control the central and periphery nervous systems. Serotonin (5-HT), in particular, is a versatile chemical messenger responsible for a multitude of biological processes, such as regulation of emotion, vasoconstriction, and bone metabolism. The study of serotonergic complex pathways is vital and essential in drug discovery for the diseases that result from the depletion and deregulation of serotonin in synapse. The extracellular concentration of serotonin is controlled by several transporters, most preferably the serotonin transporter (SERT). Selective serotonin reuptake inhibitors (SSRIs), along with dual- and triple-acting inhibitors, affect SERT and hence 5-HT in depression and related diseases. In this present investigation, firstly, a set of fluorescent analogs of neurotransmitter probes based on ethylamino-functionalized substrates were successfully designed and these fluorescent probes were synthesized by convenient synthetic methods. Secondly, optical properties of these fluorescent probes were investigated in organic medium, in order to test their suitability for screening and imaging the biological cells. Finally, their uptake was examined in the murine osteocytic cell line, MLO-Y4, platelets of blood sample and HEK-293 cells expressing the dopamine transporter (DAT), norepinephrine transporter (NET) or SERT. The fluorescent probes targeting bone-derived cell line expressing 5-HTT provide useful information in understanding the dynamics of 5-HT regulation with respect to SSRI treatment. A novel fluorescent analog of 5-HT probe was developed that may be utilized to study 5-HTT function in the context of 5-HT uptake or regulation in cell culture, tissue explants, or even in vivo.

Thesis (Ph. D.)--University of Nevada, Reno, 2005. / "May, 2005." Includes bibliographical references. Online version available on the World Wide Web. Library also has microfilm. Ann Arbor, Mich. : ProQuest Information and Learning Company, [2005]. 1 microfilm reel ; 35 mm.

The generation and propagation of fluorescence light within biological tissue offers the potential for biomedical diagnostics and analyte sensing. Arising from an exogenous fluorescent dye injected as a contrast agent or immobilized in a polymer implant, the fluorescent decay kinetics can be sensitive to the tissue??s biochemical environment, providing quantitative in vivo information of the confined tissue site. The impact of light propagation and decay kinetics upon the measured signals is important for consideration, simply because tissue scatters light, giving rise to nanosecond photon time-of-flights that are comparable to fluorescence relaxation kinetics. The goal of this study is to develop a time-dependent model describing (i) the generation of fluorescence from dyes exhibiting multi-exponential or more complex kinetics and (ii) its propagation in scattering media. In the preliminary study, fluorescence lifetime spectroscopy is investigated in tissue-like scattering solution. Two fluorescent dyes, 3,3-diethylthiatricarbocyanine iodide (DTTCI) and Indocynanine Green (ICG), which exhibit distinctly different lifetimes and each exhibits single-exponential decay kinetics, were employed. Measurements of phase-modulation as a function of modulation frequency were made at varying concentration ratios of the two dyes to experimentally simulate fluorescence multi-exponential decay kinetics in non-scattering and scattering solutions. The results suggest that frequency-domain measurements of fluorescent decay kinetics along with models of light propagation may be enhanced by scatter in order to probe kinetics more sensitively than in non-scattering solutions. The next study involved fluorescence lifetime sensing in scattering and non-scattering solutions with a pH sensitive dye, Carboxy Seminaphthofluorescein-1 (C-SNAFL-1), which is known to exhibit multi-exponential decay kinetics. The results demonstrate accurate pH sensing in scattering solution via fluorescence kinetics using a simplified propagation model incorporating an average lifetime. Finally, fluorescence lifetime sensing in immobilized systems were investigated. C-SNAFL-1 was immobilized in poly(ethylene glycol) (PEG) microparticles that were immersed in buffered polystyrene solutions. The results demonstrate the ability to perform pH sensing with fluorescence lifetime without the confounding effect of fluorophore loading or the use of 'reference' measurement within multiply scattering systems. In addition, the stability of the immobilized fluorescence sensor and the reliability of fluorescence lifetime measurement verify the prospect of this technology for implantable purposes.

In these days, nanomaterials are applied in a variety of biomedical applications including magnetic resonance imaging (MRI), cell imaging, drug delivery, and cell separation. Most MRI contrast agents affect the longitudinal relaxation time (T1) and transverse relaxation time (T2) of water protons in the tissue and result in increased positive or negative contrast. Here, we report the optimization of r1 (1/T1) or r2 (1/T2) relaxivity dynamics with diameter controlled gadolinium oxide nanocrystals (2~22 nm) and iron based magnetic nanocrystals (4 ~33 nm). The r1 and r2 MR relaxivity values of hydrated nanocrystals were optimized and examined depending on their core diameter, surface coating, and compositions; the high r1 value of gadolinium oxide was 40-60 S-1mM-1, which is 10-15 fold higher than that of commercial Gd (III) chelates (4.3~4.6 S-1mM-1). Moreover, in vitro toxicological studies revealed that polymer coated nanocrystals suspensions had no significant effect on human dermal fibroblast (HDF) cells even at high concentration. Towards multimodal imaging or multifunctional ability, we developed the iron oxide/QDs complexes, which consist of cores of iron oxide that act as nucleation sites for fluorescent QDs. The choice of variable QDs helped to visualize and remove large iron oxide materials in a magnetic separation. Additionally, diluted materials concentrated on the magnet could be fluorescently detected even at very low concentration. The designed MRI or multifunctional nanomaterials will give great and powerful uses in biomedical applications.

The challenges in modern biological imaging applications are two-fold: (i) to develop better methods of imaging, and (ii) develop dyes that are suitable for these methods. This dissertation deals with the design and synthesis of dyes mainly by modification of known dyes to make them suitable for modern biological applications. Towards this aim, novel ways of derivatizing BODIPY dyes are explored. One method involves extending the conjugation via phenyl acetylene units, pushing fluorescence wavelengths near 600 nm. A different approach deals with C-H functionalization of BODIPY in which the fluors are functionalized with acrylate units, extending their fluorescence to the red. The BODIPY dyes developed are then incorporated in through-bond energy transfer cassettes. We examine the factors affecting energy transfer efficiencies by synthesizing analogs of the cassettes and also studying the electrochemical behavior of the donor and acceptor parts. The concept of through-bond energy transfer is incorporated into conjugated polymers by random incorporation of BODIPY dyes into polyfluorenes. The ideal ratio of fluorene to BODIPY parts was found to be 4:1. The BODIPY doping agents result in dispersed emissions when excited the polyfluorene polymers. Concurrently, the polyfluorene backbone acts as an energy harvester for the BODIPY dyes, in effect increasing their molar absorptivities. Finally the use of BODIPY dyes as photodynamic therapeutic agents was examined. We found that BODIPY dyes are efficient at producing reactive oxygen species when halogens are attached directly on the BODIPY core. Furthermore, the mechanism of cell death by using such agents was elucidated. Attachment of the most promising agent to polyglutamic acid is done to promote the EPR effect. Lastly we develop a potentially new type of PDT agent that absorbs strongly above 800 nm, permitting its use in deep tissue PDT.

Polyfluorene (PF) and its derivates as well-known fluorescent materials are promising materials in optoelectronic applications due to their high quantum yields in the solid state. Nevertheless, the easy chain inter-action in PFs to result in the unfavorable associations (aggregate and excimer) are generally considered to be detrimental to the emission efficiency in the concentrated solid state and/or at high temperatures. In the study, restraining the extent of associations is therefore by embedding fluorene-based alternative and block copolymers in crosslinked network as matrix. Firstly, alternative copolymers with fluorene connected by anthracene (or pyridine or fluorene) ring were prepared through Suzuki coupling. In this way, the steric hindrance between the o-hydrogens in the neighboring aromatic ring causes the twisting of the constructed polymer chain and the resulting twisting chain conformation keeps the polymer chains from the unfavorable inter-chain interactions and reduces the extent of the association. Secondly, the alternative fluorene-anthracene copolymer (a-PFA) from the first approach can be further chemically formulated to obtain a triblock copolymer with the central a-PFA rod block connected by two flexible poly(methyl methacrylate) (PMMA) segments. In this way, the two flexible PMMA chains serve as spatial isolator to keep the central PFA rod from approaching each other and a reduced extent of association is expected for this block polymer of b-PMMA-PFA. Thirdly, all the alternative and block copolymers cited above were immersed in the curable liquid methyl metharcylate (MMA)/ditrimethylolproanetetracrylate (DTTPT) monomer mixtures and photo-irradiated to obtain composites with the fluorescent polymers immersed in the cured crosslinked network. The chain morphology and thus the degree of associations will be successfully frozen by the immobilized crosslinked network. For systems before and after photo-irradiation, the degree of aggregation was evaluated by Uv-vis absorption, photoluminescent (PL) and PL excitation spectroscopy. Polymer concentration was found to be important factor in controlling the degree of aggregation and was discussed in this study. In addition, the cured solid composites after high-temperature annealing were studied and compared with pure PFs to evaluate the effectiveness of this crosslinking strategy in restraining the extent of aggregation. In most cases, quantum yields (£XPLs) also were measured to evaluate the effectiveness of this strategy.

The generation and propagation of fluorescence light within biological tissue offers the potential for biomedical diagnostics and analyte sensing. Arising from an exogenous fluorescent dye injected as a contrast agent or immobilized in a polymer implant, the fluorescent decay kinetics can be sensitive to the tissue??s biochemical environment, providing quantitative in vivo information of the confined tissue site. The impact of light propagation and decay kinetics upon the measured signals is important for consideration, simply because tissue scatters light, giving rise to nanosecond photon time-of-flights that are comparable to fluorescence relaxation kinetics. The goal of this study is to develop a time-dependent model describing (i) the generation of fluorescence from dyes exhibiting multi-exponential or more complex kinetics and (ii) its propagation in scattering media. In the preliminary study, fluorescence lifetime spectroscopy is investigated in tissue-like scattering solution. Two fluorescent dyes, 3,3-diethylthiatricarbocyanine iodide (DTTCI) and Indocynanine Green (ICG), which exhibit distinctly different lifetimes and each exhibits single-exponential decay kinetics, were employed. Measurements of phase-modulation as a function of modulation frequency were made at varying concentration ratios of the two dyes to experimentally simulate fluorescence multi-exponential decay kinetics in non-scattering and scattering solutions. The results suggest that frequency-domain measurements of fluorescent decay kinetics along with models of light propagation may be enhanced by scatter in order to probe kinetics more sensitively than in non-scattering solutions. The next study involved fluorescence lifetime sensing in scattering and non-scattering solutions with a pH sensitive dye, Carboxy Seminaphthofluorescein-1 (C-SNAFL-1), which is known to exhibit multi-exponential decay kinetics. The results demonstrate accurate pH sensing in scattering solution via fluorescence kinetics using a simplified propagation model incorporating an average lifetime. Finally, fluorescence lifetime sensing in immobilized systems were investigated. C-SNAFL-1 was immobilized in poly(ethylene glycol) (PEG) microparticles that were immersed in buffered polystyrene solutions. The results demonstrate the ability to perform pH sensing with fluorescence lifetime without the confounding effect of fluorophore loading or the use of 'reference' measurement within multiply scattering systems. In addition, the stability of the immobilized fluorescence sensor and the reliability of fluorescence lifetime measurement verify the prospect of this technology for implantable purposes.